A T-stem slip in human mitochondrial tRNALeu(CUN) governs its charging capacity

State Key Laboratory of Molecular Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences 320 Yue Yang Road, Shanghai 200031, People's Republic of China.
Nucleic Acids Research (Impact Factor: 9.11). 02/2005; 33(11):3606-13. DOI: 10.1093/nar/gki677
Source: PubMed


The human mitochondrial tRNALeu(CUN) [hmtRNALeu(CUN)] corresponds to the most abundant codon for leucine in human mitochondrial protein genes. Here, in vitro studies reveal that the U48C substitution in hmtRNALeu(CUN), which corresponds to the pathological T12311C gene mutation, improved the aminoacylation efficiency of hmtRNALeu(CUN). Enzymatic probing suggested a more flexible secondary structure in the wild-type hmtRNALeu(CUN) transcript compared with the U48C mutant. Structural analysis revealed that the flexibility of hmtRNALeu(CUN) facilitates a T-stem slip resulting in two potential tertiary structures. Several rationally designed tRNALeu(CUN) mutants were generated to examine the structural and functional consequences of the T-stem slip. Examination of these
hmtRNALeu(CUN) mutants indicated that the T-stem slip governs tRNA accepting activity. These results suggest a novel, self-regulation
mechanism of tRNA structure and function.

Download full-text


Available from: Rui Hao, May 06, 2014
  • Source
    • "The increased levels of aminoacylated tRNAHis in mutant cell lines may be due to the instability of the mutant tRNA, where aminoacylation may provide some level of stabilization by compensatory effect (36,37). Alternatively, the mutant tRNAHis improperly charged by cognate amino acid(s) may contribute to the increasing aminoacylation level of tRNAHis (38). A failure to aminoacylate tRNA properly then makes the mutant tRNAHis to be metabolically less stable and more subject to degradation, thereby lowering the level of the tRNA, as in the case of 3243A>G mutation in the tRNALeu(UUR) (25,39). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In this report, we investigated the molecular genetic mechanism underlying the deafness-associated mitochondrial tRNAHis 12201T>C mutation. The destabilization of a highly conserved base-pairing (5A-68U) by the m.12201T>C mutation alters structure and function of tRNAHis. Using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mtDNA-less (ρo) cells, we showed ∼70% decrease in the steady-state level of tRNAHis in mutant cybrids, compared with control cybrids. The mutation changed the conformation of tRNAHis, as suggested by slower electrophoretic mobility of mutated tRNA with respect to the wild-type molecule. However, ∼60% increase in aminoacylated level of tRNAHis was observed in mutant cells. The failure in tRNAHis metabolism was responsible for the variable reductions in seven mtDNA-encoded polypeptides in mutant cells, ranging from 37 to 81%, with the average of ∼46% reduction, as compared with those of control cells. The impaired mitochondrial translation caused defects in respiratory capacity in mutant cells. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increase in the production of reactive oxygen species in the mutant cells. The data provide the evidence for a mitochondrial tRNAHis mutation leading to deafness.
    Full-text · Article · Jun 2014 · Nucleic Acids Research
  • Source
    • "Our laboratory has investigated the interaction between LeuRS and tRNALeu from various species, including Escherichia coli, Aquifex aeolicus, Giardia lamblia and human (cytosolic and mitochondrial) (6,8–11). The above-mentioned tRNALeu could be purified after overexpression in E. coli or transcribed by T7 RNA polymerase in vitro. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNA(Leu) to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNA(Leu) knockout strains carrying deletions of the genes for tRNA(Leu)(GAG) and tRNA(Leu)(UAG). Disrupting the single gene encoding tRNA(Leu)(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNA(Leu)(UAG) had a lethal effect on the yeast strain, indicating that tRNA(Leu)(UAG) decoding capacity could not be compensated by another tRNA(Leu) isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNA(Leu)(UAG), a selection to identify critical tRNA(Leu) elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA.
    Full-text · Article · Aug 2012 · Nucleic Acids Research
  • Source
    • "The model of TtLeuRS with an MmLinker in place of the CP1 domain was on the basis of the crystal structure of TtLeuRS (PDB code 2BYT)[20]. The tRNA docking model of TtLeuRS with the 3 end of tRNA in the synthetic active site was on the basis of the structure of the complex of PhLeuRS (Pyrococcus horikoshii LeuRS) and tRNA (PDB code 1WZ2)[21]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.
    Preview · Article · Feb 2012 · Biochemical Journal
Show more